PROJECT SUMMARY PROJECT 5 ? CIRCADIAN RHYTHMS Circadian rhythm and sleep disruptions increase the vulnerability for addiction disorders, though the genetics and molecular mechanisms underlying this association remain poorly understood. Genome-wide and more targeted single-nucleotide polymorphism (SNP) association studies have revealed significant associations between circadian gene SNPs and addiction disorders. While circadian mutant mice (single gene mutation) have been useful for beginning to understand these mechanisms, recent genome-wide approaches strongly indicate that there are many genes that display circadian oscillation and/or influence the robustness of the molecular clock. Therefore, a number of genes may have novel roles in the regulation of the molecular clock and addiction related behaviors. An ideal biological tool for investigating the genetics underlying the relationships between circadian rhythms and addiction related phenotypes are the Collaborative Cross (CC) and the Diversity Outbred (DO) mouse populations which are bred and housed at Jackson Labs (JAX). The DO genome consists of ~45 million unique polymorphisms and allelic combinations, and several million structural variants, which drives extensive genetic and phenotypic variation. This highly diverse genome is a powerful system for relatively fast, cost-effective, and high-precision genetic analyses to identify novel genetic loci underlying complex addiction phenotypes. This project (Project 5) will take advantage of mouse cohorts 1, 2 and 3 described in the center, and from behavioral data generated by the Behavioral Phenotyping Core associated with projects led by Drs. Jentsch, Chesler, Damaj and Tarantino. These projects will investigate the genetics underlying behavioral impulsivity, nicotine preference, cocaine sensitization and cocaine self- administration. We will collect tissue samples from all mice for which behavior is recorded, isolate fibroblasts and use these cells to measure molecular rhythms across multiple days. We will also determine if genetic variations in the CC/DO founder strains and CC lead to changes in mouse circadian behavior and include these data along with the molecular data in a large center-wide correlation, which will take place in the Integrative Genetics and Genomics Core at JAX. Fibroblasts from DO mice will be used to identify candidate genetic loci, and we will utilize the Mouse Resources and Validation Core to provide us with iPSC-derived neurons and mice with specific genetic variations for validation studies. Finally, we will measure rhythms in core clock genes, input and output gene expression, as well as markers of epigenetic gene regulation in the suprachiasmatic nucleus (SCN) and other addiction-related brain regions such as the nucleus accumbens (NAc), ventral tegmental area (VTA) and prefrontal cortex (PFC) in the CC/DO founder lines and ultimately in variants identified in our large DO screen. Through this unique and collaborative project we will identify common phenotypes and ultimately genes that associate with both circadian rhythm disruptions and addiction- vulnerability.